1. Field of the Invention
The invention relates to countercurrent chromatography systems, and more particularly to an improved instrument design for use in countercurrent chromatography.
2. Description of the Related Art
Chromatography is a separation process that is achieved by distributing the substances to be separated between a mobile phase and a stationary phase. Those substances distributed preferentially in the moving phase pass through the chromatographic system faster than those that are distributed preferentially in the stationary phase. As a consequence, the substances are eluted from the column in inverse order of their distribution coefficients with respect to the stationary phase.
Chromatography is widely used for the separation, identification, and determination of the chemical components in a complex mixture. Chromatographic separation can be utilized to separate gases, volatile substances, nonvolatile material, polymeric material, and a wide variety of organic and biological substances.
The performance of countercurrent chromatography systems depends largely on the amount of stationary phase retained in the column, which determines both the resolving power of the solute peaks and the sample loading capacity. Numerous countercurrent chromatography systems have been developed to optimize the retention of the stationary phase of a sample in the column. The maximum attainable retention level tends to fall sharply with the application of higher flow rates of the mobile phase, resulting in loss of peak resolution. Consequently, the applicable flow rate has become one of the major limiting factors in countercurrent chromatography.
Some countercurrent chromatography systems utilize a complex hydrodynamic motion in two solvent phases within a column comprising a rotating coiled tube. If, for example, a horizontally mounted coil is filled with water and is rotated around its own axis, any object, either heavier or lighter than the water present in the column will tend to move toward one end of the coil. This end is then called the “head” and the other end, the “tail” of the coil.
When the coil is filled with two immiscible solvent phases, the rotation establishes a hydrodynamic equilibrium between the two solvent phases, where the two phases are distributed in each turn at a given volume ratio (equilibrium volume ratio) and any excess of either phase remains at the respective tail of the coil for each solvent.
When the coil is filled with one of the solvents as a stationary phase and the other solvent is eluted from the coil from its head end, the hydrodynamic equilibrium tends to maintain the original equilibrium volume ratio of the two phases in the coil and thereby a certain volume of the stationary phase is permanently retained in the coil while the two phases are undergoing vigorous agitation with rotation of the coil. As a result, the sample solutes present in one phase and introduced locally at the inlet of the coil are subjected to an efficient partition process between the two phases and are chromatographically separated according to their partition coefficients.
In some cases, countercurrent chromatography utilizes a multi-layer coil as a separation column to produce a high efficiency separation with relatively favorable retention of the stationary phase in many solvent systems. Thus, countercurrent chromatography has been employed to achieve efficient separation of compounds in a sample solution under relatively high flow rates.
A structure that can be used in a countercurrent chromatography column assembly comprises a plurality of separation disks having a plurality of spiral flow channels carved, etched, or molded on the surface of a first side of each separation disk as described in U.S. Pat. No. 6,379,973, for example. The spiral flow channel has an inlet end and an outlet end, wherein fluid typically flows along the path of the spiral channel from the inlet end to the outlet end. The spiral channel of one separation disk can be serially connected to the spiral channel of another separation disk by stacking multiple separation disks adjacent to one another with a septum separating each pair. Preferably, an outlet end of a channel on one disk connects to the inlet end of the channel on the next adjacent disk.
An alternative structure that can be used in a countercurrent chromatography column assembly is described in international Patent Publication WO/2004/085020, wherein the column is formed as a length of tubing which is installed within one or more grooves in a plate or disk (also referred to herein as a tube support).
One embodiment of such a plate or disk shaped tube support for use in a countercurrent chromatography apparatus is illustrated in FIGS. 1 and 2. FIG. 1A is a top view, showing the upper surface, of the tube support. FIG. 1B is a cross-sectional view of the tube support taken along the line B-B of FIG. 1A. FIG. 1C shows the lower surface of the tube support.
In this embodiment, four spiral grooves 22, 24, 26, 28 are etched into the upper surface of the tube support. Additionally, four return paths 32, 34, 36, 38 are etched into the lower surface of the tube support. Notches 42, 44, 46, 48 are etched into the tube support proximate to locations O1, O2, O3, and O4, to allow for tubing to wrap around the tube support from the upper surface to the lower surface. Similarly, notches 52, 54, 56, 58 are etched into the tube support proximate to locations I1, I2, I3, and I4 to allow for tubing to wrap around the tube support from the lower surface to the upper surface.
In operation, tubing 50 is placed within the grooves of the tube support, winding on the upper surface from I1 to O1, then returning via a return path on the lower surface of the tube support from O1 to I2. FIG. 2 shows the cross-sectional view of the tube support of FIG. 1, with tubing in place.
As vertical portions of tubing 55 do nothing to contribute to countercurrent chromatography, the length of tubing winding between the upper surface and lower surface via the notches is wasted. Additionally, as many layers of tubing are placed within the tube support, the notches may soon fill with tubing, limiting the number of spirals of tubing which can be placed in the tube support. Furthermore, the requirement to etch a pattern on both sides of the disk or plate, and the necessity of the notches, increases the cost of manufacturing.